Search

Background The status of the tumor microenvironment (TME) can profoundly affect the response to immune-based therapies for the treatment of cancer. Results previously reported from the AWARE-1 study in early breast cancer patients demonstrated that pelareorep (pela, an oncolytic reovirus), alone or in combination with checkpoint inhibitor (CPI) therapy, modified the inflammatory state of the TME. We also showed that many of pela’s effects on the TME were enhanced by the addition of checkpoint blockade. Here, we report the effect of treatment with pela on selected molecular markers associated with enhanced anti-tumor immunity. Methods Newly diagnosed HR+/HER2- early BC patients were enrolled into two cohorts: Cohort 1 (C1): pela + letrozole (n=10); and Cohort 2 (C2): pela + letrozole + atezolizumab (n=10). Pela was intravenously administered on days 1, 2 and 8, 9, and atezolizumab was given on day 3. For this analysis, tumor biopsies (FFPE samples) collected pre-treatment (D1) and on days 3 (D3, prior to the atezolizumab administration) were examined by GeoMx digital spatial profiling (DSP, using Nanostring’s Cancer Transcriptome Atlas [CTA]). Moreover, the expression of 770 immune-related genes was analyzed using a specific immune panel (n=20). Gene Set Enrichment Analysis (GSEA) (version 4.1.0) was used to assess pela-induced activation pathways. Results GeoMx DSP showed that pela therapy significantly activated IFN-gamma signaling and associated interferon response genes from D1 to D3 (Normalized Enrichment Score [NES] = 3.3, p-values < 0.02) in the cytokeratin-positive subset of the tumor samples. GSEA of the immune dataset (730 immune genes + 30 housekeeping genes) from the whole tissue also showed a significant upregulation of IFN-gamma signaling pathway genes (FDR < 25%, p-value< 0.001). Increases were also observed in genes reported to be associated with an enhanced tumor inflammatory signal (TIS) including PD-L1, IDO1, HLA-E and STAT1 (p-values < 0.005). Conclusions These results demonstrate that treatment with pela alters the TME to induce and enhance anti-tumor immunity. This enhancement of anti-tumor immunity may potentiate the TME for CPI therapy. Citation Format: Houra Loghmani, Joaquín Gavilá, Luis Manso, Matt Coffey, Richard Trauger, Fernando Salvador, Tomás Pascual, Aleix Prat, Thomas Heineman. PD4-03 Pelareorep primes the tumor for checkpoint inhibition therapy by activating the interferon-gamma signaling pathway and tumor inflammation signature in early breast cancer patients - results of the AWARE-1 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-03.

Background: Multiple myeloma (MM) is an incurable cancer characterized by clonal plasma cell proliferation in the bone marrow, accounting for approximately 10% of all hematologic malignancies. Recently, patients with relapsed or refractory disease have been treated with a combination of the oncolytic reovirus Pelareorep, bortezomib, and dexamethasone, which was well-tolerated and led to prolonged progression free survival of over 3 years in a subset of patients. To understand the complex tumor immune microenvironment (TiME) and immune responses in patients before and after this treatment, we used imaging mass cytometry (IMC) to perform single cell, highly multiplexed, analysis of these patients’ bone marrow samples. Methods: We comprehensively characterized the changes in the MM TIME in pre and post bone marrow biopsy specimens taken from patients treated on a Phase 1b study with a combination of Pelareorep, bortezomib, and dexamethasone. For analysis with IMC, a marker panel of 35 antibodies was assembled to interrogate the various immune subsets of the bone marrow biopsies; each of these antibodies were conjugated to a unique metal isotope. After validation, the antibody cocktail was used to stain the biopsies. Pixel-based classification was performed in Ilastik to generate cell probability masks and processed in Cellprofiler. PhenoGraph was run in HistoCAT to identify the unique phenotypes. Rstudio was used for t-stochastic neighborhood embedding (tSNE) plot generation, and nearest neighbor analyses. ImaCytE was used for image visualization and spatial analysis. Results: Initial visualization of the raw, unsegmented data showed increased infiltration of natural killer cells and T cells in the post-treatment samples when compared against the pre-treatment samples. These changes correlated with immunohistochemical findings, clinical response to treatment, and changes in T cell clonality. After segmentation, the marker expression heatmaps for each of the clusters identified by PhenoGraph and the further subphenotyping in Rstudio showed complex ecosystems of cell-cell interactions. Nearest neighbor spatial analysis of the post-treatment samples revealed that NK cells (NKG2D+ and NKG2A+ subsets), monocytes (CD14+), macrophages (CD68+), cytotoxic T cells (CD3+, CD8+), and T helper cells (CD3+, CD4+) were significantly closer to the Pelareorep-primed MM than the non-primed MM. Further analysis in ImaCytE highlighted specific instances of these immune neighborhoods. Conclusions: IMC allows us to analyze the potent immune response and cellular interactions in the tumor microenvironment in multiple myeloma treated with Pelareorep and Bortezomib. Characterization of these complex interactions allows for a deeper understanding of the key mechanisms of action of these treatments and planning of future combination studies. Citation Format: Julian Olea, Kaijin Wu, Anthony Colombo, Claudia Villa Celi, Thomas Heineman, Matt Coffey, Steffan T. Nawrocki, Akil Merchant, Kevin R. Kelly. Using imaging mass cytometry to visualize the multiple myeloma tumor microenvironment post immune priming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6354.

Background: Pelareorep is an immuno-oncolytic virus that induces an inflamed tumor phenotype in metastatic adenocarcinoma of the pancreas (MAP). Systemically delivered pelareorep in combination with chemotherapy achieves 1 & 2 year-survival rates of 46% & 24% in MAP pts, respectively. Analysis of tumor tissue from patients (pts) treated with pelareorep, chemotherapy and anti-PD-L1 have shown reovirus RNA and protein replication, T-cell infiltration, and upregulation of PD-L1, highlighting that effective T-cell recognition of tumor antigens may be critical to success for this combination therapy. Thus, we hypothesized that pelareorep in combination with chemo and pembrolizumab in pts with MAP would alter the peripheral T-cell repertoire, creating new T-cell clones via the release of novel neoantigens in addition to expanding existing T-cell clones. Methods: A phase 1b study enrolled 11 MAP pts who progressed after first-line treatment. Pts received pelareorep (4.5e10 TCID 50 IV, D1 & D2), plus pembrolizumab (2mg/kg IV, D8) plus either 1) 5-FU (LV (200 mg/m2 /5-FU 200 mg /m2 IV bolus, 5-FU 1200mg/m2 continuous IV infusion D1) or 2) gemcitabine (1000 mg/m2 IV, D1), or 3) irinotecan (125 mg/m2 IV, D1) q3w, until disease progression/unacceptable toxicity. DNA from peripheral blood mononuclear cells from nine patients at cycle 1 day 1 (C1D1) & C2D1 (approx. 3 weeks later) was analyzed using the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle) sequencing the T-cell receptor beta chain region to interrogate changes in the T-cell repertoire. Results: The median Morisita index between C2D1 and C1D1 was 0.83 with 3 samples below 0.6, indicative of significant peripheral repertoire turnover. The median number of expanded clones equated to 45.7 per 100,000 cumulative templates; normal variation over 4 weeks is ~ 5-10 expanded clones. Strikingly, most (median: 86%) peripheral clonal expansion occurred in clones below the limit of detection at C1D1. Cox regression analysis revealed that high peripheral clonality correlates with progression free survival at C1D1 (p=0.01, HR=0.053). Moreover, high clonality correlates with overall survival at both C1D1 (p=0.013, HR=0.124) and C2D1 (p=0.010, HR=0.079). Conclusions: High levels of peripheral T-cell repertoire turnover occur between C1D1 and C2D1. Repertoire turnover is accompanied by significant clonal expansion, mostly by expansion of new clones (i.e. undetected in C1D1). Higher peripheral clonality is associated with better progression free survival at C1D1, and overall survival at C1D1 and C2D1. This research highlights the potential utility of T-cell clonality as a predictive and prognostic biomarker to pelareorep therapy and warrants further clinical investigation. Citation Format: Grey A. Wilkinson, Devalingam Mahalingam, Sukeshi Patel Arora, Paul A. Fields, Patrick Raber, Karol Cheetham, Matt Coffey. Exploratory analysis of T cell repertoire dynamics upon systemic treatment with the oncolytic virus pelareorep in combination with pembrolizumab and chemotherapy in patients with advanced pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2272.